This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION: In a revision of an application, Dr. Khan proposes, as previously, to investigate sensory excitation and signal amplification in the chemotactic system of Escherichia coli using a rapid assay for measurement of chemotactic excitation that he has developed and that involves flash photo release of caged compounds and computer-assisted video analysis of cellular behavior. The overall goal is to understand signal amplification in terms of the intracellular phosphorylation cascade mediating signalling between the chemoreceptors and the flagellar motor. Photorelease of caged attractants (serine and aspartate) or repellents (protons and leucine) will be used to apply stimuli to free swimming cells at defined times and thus allow measurement of the speed and amplitude of the excitation response. Initial studies using caged serine have been recently published. This work will be extended to investigate response to small-step changes in stimulant concentration at different initial concentrations, and to investigate responses in cells with altered initial swim/tumble balances as the result of metabolic and mutational perturbations. Dr. Khan also proposes to continue work on the development of a computer program for automated analysis of a field of tethered cells. This would allow his proposed analyses of responses of swimming cells, representing a summation of action of the multiple flagella on each cell, to be compared to response of tethered cells, representing the action of a single flagellum. A second aim is to relate the kinetics of phosphotransfer in vitro to the kinetics of excitation in intact cells by examining excitation in mutants defective in interaction between CheY and the flagellar motor, in strains containing increased amounts of CheY or CheY plus CheZ or CheB, and in mutants defective in CheZ or in phosphotransfer between CheA and CheY. A third aim is to characterize the affects of the adaptation components on excitation by examining excitation in mutants altered in the CheB methylesterase or in the methyl-accepting site of a chemoreceptor. The fourth aim is to develop and characterize electroporation techniques that will allow dependable and informative experiments using caged compounds introduced into cells.